Background
The development of effective antimicrobial agents was once seen as a definitive cure for bacterial diseases. But even before the development of the first antibiotics, bacteria had demonstrated an ability to adapt to stress in the environment, resulting in the development of resistance. In recent years, the variety of antimicrobial agents has increased substantially, along with a parallel increase in resistant pathogenic microorganisms. Resistance is now recognized against all clinically available antimicrobial agents. The response to antimicrobial resistance in the medical community has been to use new or alternative antibiotics not previously used against the resistant bacteria. This approach has required the continuous development of new antibiotics, either as modifications of currently existing compounds or as combinations of compounds that may inhibit or bypass the bacterial resistance mechanisms.
Natural polycationic antibiotic peptides have been found in many different species of animals and insects and shown to have broad antimicrobial activity. In mammals, these antimicrobial peptides are mainly represented by two families, the defensins and the cathelicidins. Nearly all of these peptides have membrane affinity, and can permeate and permeabilize bacterial membranes, resulting in injury, lysis, and/or death to the microbes. For example, the human peptides termed alpha-defensins are produced by neutrophils and intestinal Paneth cells. In three-dimensions, defensins manifest an amphiphilic, largely beta-sheet structure, with a polar face formed largely by its arginines and with N- and C-terminal residues playing an important role in defining antimicrobial potency and spectrum. (See Gudmundsson et al. (1999) J Immunol Methods 232(1-2):45-54.) Antimicrobial peptides are reviewed by Hancock and Lehrer (1998) Trends in Biotechnology 16:82.
Cystic fibrosis (CF) is an inherited disorder that occurs in one of every 3,300 U.S. newborns. It affects some 30,000 Americans today. The median survival age of patients with CF is only 31.3 years, making CF the most common life-shortening inherited disease in the U.S. Most CF patients die from pulmonary failure that results from chronic, progressive infection by Pseudomonas aeruginosaxe2x80x94a Gram-negative bacterium that is widely distributed throughout the environment. P. aeruginosa has limited ability to infect normal individuals, but can be a devastating secondary invader in immunocompromised, severely burned, or antibiotic-treated persons. Because Pseudomonas aeruginosa strains frequently are or become resistant to conventional antibiotics, infections caused by them are often difficult to eradicate.
The in vitro activity of antimicrobial peptides, including cecropin P1, indolicidin, magainin II, nisin and ranalexin has been tested against clinical isolates of P. aeruginosa. The peptides were found to have a varied range of inhibitory values, and showed some synergy when combined with conventional antibiotics (Giacometti et al (1999) J Antimicrob Chemother. 44(5):641-5)
There is a clinical need for novel antibiotic agents that are active against drug resistant Gram-negative bacteria, and which have low toxicity against mammalian cells. The present invention addresses this need.
Relevant Literature
Saiman et al. (1999) Pediatr Pulmonol, Suppl. 17:320 report that drug resistant organisms from CF patients are inhibited by cathelicidin peptides; and Brogden et al. (1999) Pediatr Pulmonol, Suppl. 17:320 report on the efficacy of SMAP29 in an ovine model of pulmonary infection and its potential for treating P. aeruginosa infection in patients with cystic fibrosis (CF).
Methods and compositions are provided for the use of novispirin peptides. Novispirin peptides are small antimicrobial agents with potent activity against Gram-negative bacteria, including Chlamydia trachomatis, Pseudomonas aeruginosa, Eschelichia coli and Stenotrophomonas maltophilia. The peptides are nonhemolytic, exhibit reduced in vitro cytotoxicity relative to other antimicrobial peptides, and are well-tolerated in vivo after intravenous injection. Novispirins are equally effective against growing and stationary phase P. aeruginosa, and they retain activity in the presence of high concentrations of salt or human serum. Novispirins also bind lipopolysaccharide (LPS), a property that may mitigate symptoms associated with Gram-negative bacterial infection.
A pharmaceutical composition comprising novispirin as an active agent is administered to a patient suffering from a microbial infection, particularly bacterial infections. The protein is also effective at killing a variety of microbial organisms in vitro. Novispirin may be administered alone, or in combination with other bacteriocidal agents, e.g. antibiotics, as a cocktail of effective peptides, etc. Novispirin mediated killing of microbes is also useful for modeling and screening novel antibiotics.